The present invention relates to the use of high-gradient magnetic cell separation (HGMS) techniques in the process of modifying selected cells.
The prior art addresses various methods and techniques to modify cells outside the body. Surface staining of cells by the attachment of antibodies bound to fluorescent moieties is one frequent modification. Intracellular staining after fixation is also common. Kappa and lambda light chains can be targeted by such stains and used to assay for monoclonal tumor cell populations. While stains of this sort are frequently specific, cells can be modified in myriad non-specific ways, such as by exposure to a pharmacological or chemical agent, treatment with a hormone or other compound known to mediate cellular activity, or transfection with genes to bring about a recombinant product. Cell modification techniques thus have both research-oriented and treatment-oriented potential.
In the prior art, modifications like staining are performed either with cells in suspension or with monolayers of cells immobilized on different surfaces (such as a microscope slide), to allow the modifying agent to access all the cells equally. Staining or otherwise modifying cells in these fashions are known in the art. Recent advancements in monolayer modification are exemplified by Lebkowski, et al., Isolation, Activation, Expansion and Gene Transduction of Cell Based Theraputics Using Polysterene Immunoaffinity Devices, in Cell Separation Methods and Applications, Recktenwald, et al., eds. (1998) and in U.S. Pat. No. 5,912,177. However, these methods can be time-consuming and often involve multiple washing steps, creating a potential risk of cell loss. These methods are especially problematic where small numbers of cells are involved.
In the prior art, it is not recommended to modify cells in aggregate forms (such as the pellet formed after centrifugation). Crosslinking agents such as formaldehyde pose similar problems, as cells need to be separated from each other to prevent irreversible aggregation.
Very frequently, the practitioner will wish to modify one cell type within a heterogeneous mixture or suspension, such as lymph or blood, because subsequent to modification that specific cell type is to be studied or returned to the patient as part of a treatment or test regiment. In such circumstances, modification of cells other than the targeted class can have adverse effects. Various methods of cell separation have evolved to meet these needs.
An overview of cell separation techniques current in the art is provided by Cell Separation Methods and Applications, Recktenwald, et al., eds. (1998). One cell separation technique used involves magnetic cell separation, whereby target cells may be labeled with a magnetic marker and then selectively retained in a chamber or column exposed to a magnetic field. Kantor, et al. (1998, Magnetic Cell Sorting with Colloidal Superparamagnetic Particles, in Cell Separation Methods and Applications); Gee (1998, Immunomagnetic Cell Separation Using Antibodies and Superparamagnetic Microspheres in Cell Separation Methods and Applications); and Miltenyi, S., U.S. Pat. No. 5,411,863, disclose magnetic cell separation techniques. For high gradient magnetic separation, typically a heterogeneous suspension, containing selected cells bound to magnetic markers, is passed through a column, allowing the selected cells to adhere magnetically to the column or to a paramagnetic matrix within the column. The remainder of the suspension is eluted, leaving the selected, magnetized cells bound to the column. When the magnetic field is removed, the selected cells can be eluted.
Non-magnetic cell separation processes may be used to facilitate cell modification. These methods include immunoaffinity cell separation techniques, which are often less preferable than magnetic cell separation methods, because the former often result in increased cell loss as well as increased reagent use and salt concentration, which may require dialysis of the eluate. Lebkowski, et al. (1998, Isolation, Activation, Expansion, and Gene Transduction of Cell-Based Therapeutics Using Polystyrene Immunoaffinity Devices, in Cell Separation Methods and Applications) disclose a method whereby selected classes of peripheral blood mononuclear cells (PBMC) can be positively selected by immunoaffinity processes, in which the selected cells are immobilized by monoclonal antibodies or lectins covalently bound to polystyrene structures and then cultured, activated, or genetically modified while immobilized. A similar method for modifying stem cells is disclosed in U.S. Pat. No. 5,912,177. A central disadvantage of these methods, however, is that they bind the selected cells to a flat or otherwise two-dimensional surface, requiring a substantial surface area in relation to the number of cells to be selected. The cells must form a monolayer, as illustrated in FIG. 1. Furthermore, antibodies used for selection are affixed to the device itself and such techniques require a specific device or structure for each desired target. Moreover, releasing and resuspending selected cells from those structures can be a time consuming process.
Magnetic cell separation techniques have been used to immobilize cells as described in U.S. Pat. No. 5,622,831 and U.S. Pat. No. 5,876,593. U.S. Pat. No. 5,622,831, requires a complicated switching mechanism to release and resuspend immobilized cells and U.S. Pat. No. 5,876,593 does not employ a column, but instead requires immobilizing the cells as a substantially one-dimensional monolayer.
It would be advantageous to modify selected cells in a HGMS column, which would convey the additional advantages of the magnetic system over the immunoaffinity system, such as simple resuspension of immobilized cells, increased cell concentration, decreased reagent use, and greater effective concentration of modifying reagents.
The present invention provides methods for modifying selected cells comprising modifying magnetically labeled selected cells retained on a high gradient magnetic separation (HGMS) column thereby producing modified, selected cells. The present invention also provides methods for modifying selected cells comprising the steps of applying a population of cells to a HGMS column wherein said population of cells comprises magnetically labeled selected cells and wherein said magnetically labeled selected cells are retained by said column; and modifying said selected cells retained by said column thereby producing modified, selected cells. In other aspects, the methods further comprise removing the modified, selected cells from the column. In a further aspect, the invention comprises the additional steps of applying the removed, selected cells to a second high gradient magnetic cell separation column such that the selected cells are retained by said second column; and modifying the selected cells retained by said second column.
In other embodiments, the high gradient magnetic cell separation column contains a high volume of a matrix relative to the total volume of the column. In additional embodiments, the matrix is more than 50% of the total volume of the column and in yet further embodiments, more than 60% of the total volume of the column. In yet additional embodiments, the matrix comprises ferromagnetic spheres. In other embodiments, the removing is by removing said magnetic field.
In some embodiments, the modifying comprises intracellular staining of the selected cells; permeabilizing the selected cells; labeling the magnetically labeled selected cells with a second label; binding a biologically reactive compound to the selected cells; transfecting the selected cells with an expression vector comprising a gene of interest; applying an enzyme to the selected cells; applying a pharmacological agent to the selected cells; applying a biological or chemical agent to the selected cells; or applying multiple modifying agents. In some embodiments, the biologically reactive compound includes antibodies, ligands, proteins, peptides, nucleic acids, polynucleotides, oligonucleotides, lectins, lipids or enzymes.